Eukaryotic use of non-chimeric mutational vectors

ABSTRACT

The invention is based on the reaction of Duplex Mutational Vector in a cell-free system containing a cytoplasmic cell extract and a test plasmid. The reaction specifically converts a mutant kan r  gene to recover the resistant phenotype in transformed MutS, RecA deficient bacteria. Using this system a type of Duplex Mutational Vector termed a Non-Chimeric Mutational Vector, having no RNA:DNA hybrid-duplex is shown to be an effective substrate for eukaryotic enzymes. The invention concerns the use of Non-Chimeric Mutational Vectors protected from 3&#39; exonuclease attack in eukaryotic cells. Such protection can be conferred by replacement of a tetrathymidine linker by a nuclease resistant oligonucleotide, such as tetra-2&#39;-O-methyl-uridine, to link the two strands of the recombinagenic oligonucleobase.

1. FIELD OF THE INVENTION

Chimeraplasty concerns the introduction of directed alterations in aspecific site of the DNA of a target cell by introducing duplexoligonucleotides, which are processed by the cell's homologousrecombination and error repair systems so that the sequence of thetarget DNA is converted to that of the oligonucleotide where they aredifferent. The present invention concerns a chimeraplasty method that ispracticed in a cell-free system.

2. BACKGROUND TO THE INVENTION 2.1 Chimeraplasty

Chimeraplasty in eukaryotic cells and duplex recombinagenicoligonucleotides for use therein are disclosed in U.S. Pat. No.5,565,350, issued Oct. 15, 1996, and U.S. Pat. No. 5,731,181, issuedMar. 24, 1998 by E. B. Kmiec (collectively "Kmiec"). The recombinagenicoligonucleotides disclosed by Kmiec contained ribo-type, e.g.,2'-O-methyl-ribonucleotides, and deoxyribo-type nucleotides that werehybridized to each other and were termed Chimeric Mutational Vectors(CMV). A CMV designed to repair a mutation in the gene encodingliver/bone/kidney type alkaline phosphatase was reported in Yoon, K., etal., 1996, Proc. Natl. Acad. Sci. 93, 2071. The alkaline phosphatasegene was transiently introduced into CHO cells by a plasmid. Six hourslater the CMV was introduced. The plasmid was recovered at 24 hoursafter introduction of the CMV and analyzed. The results showed thatapproximately 30% to 38% of the alkaline phosphatase genes were repairedby the CMV.

A CMV designed to correct the mutation in the human β-globin gene thatcauses Sickle Cell Disease and its successful use was described inCole-Strauss, A., et al., 1996, Science 273, 1386. A CMV designed tocreate a mutation in a rat blood coagulation factor IX gene in thehepatocyte of a rat is disclosed in Kren et al., 1998, Nature Medicine4, 285-290. An example of a CMV having one base of a first strand thatis paired with a non-complementary base of a second strand is shown inKren et al., June 1997, Hepatology 25, 1462.

U.S. Pat. No. 5,760,012, by E. B. Kmiec, A. Cole-Strauss and K. Yoon,published as WO97/41141, Nov. 6, 1997, and U.S. Pat. No. 5,888,983,disclose methods and CMV that are useful in the treatment of geneticdiseases of hematopoietic cells, e.g., Sickle Cell Disease, Thalassemiaand Gaucher Disease.

An example of the use of a CMV having one base of a first strand that ispaired with a non-complementary base of a second strand is shown in Krenet al., June 1997, Hepatology 25, 1462. In Kren, the strand having thedifferent desired, sequence was the strand having 2'-O-methylribonucleotides, which was paired with the strand having the 3' end and5' end. U.S. Pat. No. 5,565,350 described a CMV having a single segmentof 2'-O-methylated RNA, which was located on the chain having the 5' endnucleotide.

Applicants are aware of the following provisional applications thatcontain teaching with regard to chimeric mutational vectors: By Steer etal., Ser. No. 60/045,288 filed Apr. 30, 1997; Ser. No. 60/054,837 filedAug. 5, 1997; Ser. No. 60/064,996, filed Nov. 10, 1997; and by Steer &Roy-Chowdhury et al., Serial No. 60/074,497, filed Feb. 12, 1998,entitled "Methods of Prophylaxis and Treatment by Alteration of APO Band APO E Genes."

2.2 Cell-Free Recombination

Various reports of homologous recombination using a cell-free extracthave been published.

Hotta, Y., et al., 1985, Chromosoma 93, 140-151 report the use of anextract of yeast, mouse spermatocytes and Lilium to effect homologousrecombination between two mutant pBR322 plasmids. One of the plasmidswas supercoiled, the second plasmid could be linearized or supercoiled.The maximum rate of recombination was less than 1%. A similar experimentusing mutant defective pSV2neo and extracts of EJ cells was reported inKucherlapati, R. S. et al., 1985, Molecular and Cellular Biology 5,714-720. The maximum rate of recombination was about 0.2%. Kucherlapatireported an absolute requirement that one of the mutant plasmids belinearized. In contrast Hotta, reported recombination between twocircular plasmids, although the rate of recombination between circularand linear plasmids was higher.

The report of Jessberger, R., & Berg, P., 1991, Mol. & Cell. Biol. 11,445 concerns recombination catalyzed by nuclear extracts betweenplasmids. It stands in contrast to both of the above in two respects.The rate of recombination reported was about 20%, in contrast to ratesof less than 0.5%. In addition Jessberger observed the same rate ofrecombination between circularized plasmids as between a circularizedand a linear plasmid.

A related experiment using human nuclear extracts was reported by Lopez,B. S., et al., 1992, Nucleic Acids Research 20, 501-506. Lopez reportedrecombination in a cell-free system between a linearized plasmid and anunrelated supercoiled plasmid that is not viable in the subsequentselection conditions. The linearized and supercoiled plasmid eachcontain a lacZ gene; which is a mutant in the linearized plasmid. Thelinearized plasmid is cut in the lacZ. gene at a variable distance fromthe mutation. Homologous recombination between the site of the mutationand the cut, accordingly, results in the circularization of the plasmidthat then becomes viable and the gain of lacZ function. Lopez reports nodetectable homologous recombination when the cut and the mutation were15 base pairs apart. Homologous recombination at a low level wasobserved when that distance was 27 base pairs. No further increase inthe rate of homologous recombination was observed when the distance wasmade greater than 165 base pairs. Lopez et al., 1987, Nucleic AcidsResearch

2.3 Rad51 and Rad52 Activity in Recombination

Homologous recombination is the process whereby the genes of twochromosomes are exchanged. The rate of homologous recombination betweentwo genetic loci is inversely proportional to their genetic linkage,tightly linked genes rarely recombine. In addition to its geneticfunction homologous recombination allows a somatic cell to repair DNAdamaged by double strand breaks.

The first step in homologous recombination is believed to be synapseformation. A synapse is a DNA molecule in which one chain is hybridizedto two other chains. Synapse formation requires an enzymatic activityand energy input from ATP hydrolysis. An artifactual assay in acell-free system for the enzymatic activity believed to be required forsynapse formation is "strand transfer." In a typical strand transferassay a circular single strand DNA is combined with a linear duplex toproduce a "nicked" or relaxed circular duplex and a linear singlestrand. The Rad51gene from yeast, mice and humans has been cloned andcatalyzes strand transfer. Rad51is believed to participate in synapseformation. Baumann, P., et al., 1996, Cell 87, 757-766; Gupta, R. C.,1997, Proc. Natl. Acad. Sci. 94, 463-468. The strand transfer activityis further enhanced by the presence of Rad52 protein and replicationprotein A. Baumann, P., & West, S. C., 1997, EMBO J. 16, 5198-5206; New,J. H., et al., 1998, Nature 391, 407-410; Benson, F. E., et al., 198,Nature 391, 401-404. Although RAD51 protein unlike Rec A binds to duplexDNA, Baumann & West op cit.; Benson, F. E., et al., EMBO J., 13,5764-5771, in the presence of RAD52, its binding is directed towardssingle stranded DNA.

In yeast, Rad51 or Rad52 defective individuals are radiation sensitivebecause of an inability to repair double strand breaks. In mice,Rad51knock out results in embryonic leathality. Tsuzuki, T., et al.,Proc. Natl. Acad. Sci. 93, 6236-6240; Lin, S. D., & Hasty, P. A., Mol.Cell. Biol., 16, 7133.

2.4 Cell-Free Mismatch Repair

The intrinsic (thermodynamic) fidelity of DNA replication would lead toan unacceptably high rate of mutation without the presence of an "errorcorrecting" mechanism. Mismatch repair is one such mechanism. Inmismatch repair, duplex DNA having a base paired to a non-complementarybase is processed so that one of the strands is corrected. The processinvolves the excision of one of the strands and its resynthesis. Reportsof mismatch repair in cell-free eukaryotic systems can be found inMuster-Nassal & Kolodner, 1986, Proc. Natl. Acad. Sci. 83, 7618-7622(yeast); Glazer, P. M., et al., 1987, Mol. Cell. Biol. 7, 218-224 (HeLacell); Thomas D. C., et al., 1991, J. Biol. Chem., 266, 3744-3751 (HeLacell); Holmes et al., 1991, Proc. Natl. Acad. Sci., 87, 5837-5841(HeLacell and Drosophila). The HeLa and Drosophila cell-free systems requiredthat one strand of the mismatched duplex be nicked for full activity. Bycontrast, reports of repair in Xenopus egg extracts did not require thatthe mismatched duplex be nicked. Varlet, I., et al., 1990, Proc. Natl.Acad. Sci. 87, 7883-7887. However, in Varlet the mismatch was repairedin a random fashion, i.e., the strands acted as templates with equalfrequency.

Many of the genes required for mismatch repair in yeast and humans havebeen cloned based on homology with the E. coli mismatch repair genes.Kolodner, R., 1996, Genes & Development 10, 1433-1442. Cells havingdefective mismatch repair genes show genetic instability, termedReplication Error (RER), particularly evident in microsatellite DNA, andmalignant transformation. Extracts of RER cells do not have mismatchrepair activity. Umar, A., et al., J. Biol. Chem. 269, 14367-14370.

3. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. An example of the conformation of a double hairpin typerecombinagenic oligomer. The features are: a, first strand; b, secondstrand; c, first chain of the second strand; 1,5' most nucleobase; 2,3'end nucleobase; 3,5' end nucleobase; 4,3' most nucleobase; 5, firstterminal nucleobase; 6, second terminal nucleobase.

FIG. 2. An example of the conformation of a single hairpin typerecombinagenic nucleobase with an overhang. The features are as abovewith the addition of d, the overhang. Note that the same nucleobase isboth the 5' most nucleobase of the second strand and the 5' endnucleobase.

4. SUMMARY OF THE INVENTION

Chimeraplasty is an increasingly important process for the treatment ofhuman disease and the development of useful, genetically engineeredplant and animal strains. The development of improved recombinagenicoligonucleotides has been greatly facilitated by the use of bacterialtesting systems, which give rapid and quantitative results as describedin commonly assigned regular U.S. application Ser. No. 09/078,063, filedMay 12, 1998, entitled "Non-Chimeric Mutational Vectors" by R. Kumar etal., and U.S. Provisional Application No. 60/085,191 filed May 12, 1998,entitled "Heteroduplex Mutational Vectors and Use Thereof in Bacteria"by Kumar et al., (hereafter collectively "Kumar") filed on even dateherewith, which are hereby incorporated by reference in its entirety.The techniques of Kumar do not address whether the optimalrecombinagenic oligonucleotides in bacterial systems are also optimal ineukaryotes. The prior art techniques of in vivo and cell-culturechimeraplasty are not designed for rapid quantitative analysis and areunable to utilize the same recombinagenic oligonucleobases and DNAtargets as used in the bacterial systems. Accordingly, an objective ofthe present invention is an assay that can use DNA targets andrecombinagenic oligonucleobases designed for bacterial systems torapidly evaluate the compatibility between different types ofrecombinagenic oligonucleotides and the recombination and repair enzymesof different phyla, e.g., do the recombination and mismatch repairenzymes of bacteria, plants, insects and mammals have differingsubstrate preferences?

A further objective of the invention is an assay that can rapidlydetermine whether a tissue or cell line is a target for chimeraplasty,i.e, whether it contains the requisite enzymes. A yet further objectiveis an assay to determine what agents or treatments can alter the levelof chimeraplasty activity in a cell line or tissue. A yet furtherobjective of the invention is an assay that can determine whether acompound is an agonist or antagonist of the recombination and repairpathway. An additional objective of the invention is a practical methodof making specific genetic changes in a DNA sequence in a cell-freesystem that is an alternative to polymerase chain reaction PCR-basedmethods.

The present invention meets these objectives by the unexpected discoverythat chimeraplasty can be performed in a cell-free system. Thecomponents of the cell-free system are an enzyme mixture containingstrand transfer activity and, optionally, a mismatch repair activity, atarget DNA sequence and a recombinagenic oligonucleobase. The enzymemixture can be made by obtaining a cell extract, or a mixture ofrecombinantly produced purified enzymes. The target DNA sequence ispreferably a plasmid that can be used to transform an expression hostsuch as a bacteria. In a preferred embodiment the plasmid issupercoiled. The recombinagenic oligonucleobase is any oligonucleotideor oligonucleotide derivative that can be used to introduce a sitespecific, predetermined genetic change in a cell. As used herein a DNAduplex consisting of more than 200 deoxyribonucleotides and nonucleotide derivatives is not a recombinagenic oligonucleobase.Typically, a recombinagenic oligonucleobase is characterized by being aduplex nucleotide, including nucleotide derivatives or non-nucleotideinterstrand linkers, and having between 20 and 120 nucleobases orequivalently between 10 and 60 Watson-Crick nucleobase pairs. In apreferred embodiment, the recombinagenic oligonucleobase issubstantially a duplex and contains a single 3' end and 5' end;accordingly, the strands of the duplex are covalently linked byoligonucleobase or non-oligonucleobase linkers. A further embodiment ofthe present invention is based on the discovery that the Non-ChimericMutational Vectors (NCMV), according to Kumar, are effective substratesfor the strand transfer and repair enzymes of eukaryotic and,specifically mammalian cells. Yet further embodiments of the inventionare based on the discovery that two types of recombinagenicoligonucleobases, according to Kumar, Heteroduplex Mutational Vectors(HDMV) and vectors having a single segment of ribo-type nucleobases inthe strand opposite the strand containing the 3' end nucleobase and 5'end nucleobase, unexpectedly give superior results when used witheukaryotic and specifically in mammalian strand transfer and repairenzymes. The term Duplex Mutational Vectors (DMV) is used herein torefer to CMV, HDMV and NCMV, collectively. Note that a HDMV can beeither chimeric or non-chimeric, however, the term CMV does notencompass HDMV.

5. DETAILED DESCRIPTION OF THE INVENTION

According to the present invention a reaction is carried out in areaction mixture containing an enzyme mixture comprising strand transferand mismatch repair activities, a DNA target and a recombinagenicoligonucleobase. In one embodiment the DNA target is a mutatedantibiotic resistance gene, e.g., tet or neo (kan) of a plasmid and therecombinagenic oligonucleobase is a 2'-O-methyl containing a CMVaccording to Kmiec, at about a 1:200 molar ratio. The function of themutant tet or kan is restored by specific alteration of a single base.The reaction is terminated by phenol/chloroform extraction and theextracted plasmid electroporated into RecA or MutS defective bacteria.The extent of modification of the target DNA can be determined from theratio of the recombinant (kan^(r) or tet^(r)) colonies to the parentaltype (amp^(r)). No recombinant colonies, above background, were observedwhen the plasmid and chimera were reacted separately and recombinedafter chloroform/phenol extraction. Recombinant colonies were reducedabout 90% when extracts of mismatch repair deficient cells (LoVo) wereused. These controls indicate that the modification, up to the point ofmismatch excision is completed in the reaction mixture. The frequency ofrecombinant colonies was about 5 per 10⁵ parental colonies using CMV ofthe type described in Kren et al. Nature Medicine, 1998, 4, 285-290 andCole-Strauss et al., 1996, Science 273, 1386 (a "Cole-Straus CMV").

As used herein a cell-free enzyme mixture is deemed to have strandtransfer and mismatch repair activity when the cell-free mixture can beused to obtain the above described result.

Table I below shows the effects of multiple modifications of theCole-Strauss CMV in both the bacterial and cell-free eukaryotic systems.There is a very good correlation between the activity of anymodification measured in each system. In particular the substitution of2'-O-methyl uracil for thymidine in the interstrand linkers (variants IVand V), the placement of the mutator only in the 5' strand (variant VIb)and deletion of DNA from the 3' strand significantly improved theperformance of the recombinagenic oligonucleobases in both systems.

In both systems the placement of the mutator in the 3' strand (variantVIa) resulted in a substantial loss of function to below one in 10⁵recombinant colonies. The frequency observed with variant VIa wasclearly higher than background. Accordingly, as used herein arecombinagenic oligonucleobase is an oligonucleobase of the type thatcan provide a rate of recombination in the above cell-free system atleast as high as a recombinagenic oligonucleobase made according tovariant VIa having the same mutator sequence.

Variant VII with a one base mutator sequence was observed to effectrecombination with a frequency of 4.4/10⁵. This frequency wassignificantly greater than that observed in the bacterial systems aswell as that observed in cultured cells. Without limitation as totheory, this difference is believed to be due to the relative absence ofexonucleases and endonucleases from the cell free system.

5.1 The Cell-Free Enzyme Mixture

The cell-free enzyme mixture for the practice of the invention containsthe strand transfer and the mismatch repair activities. As used hereinthe term "cell-free enzyme mixture" indicates that the mixture excludesliving cells, and preferably excludes the organelles, e.g., nuclei andmitochondria. The extent of the mismatch repair that is required in thecell-free enzyme mixture depends on the method used to detect themodification of the targeted DNA sequence and the utility.

When the modification is detected by biochemical means, e.g.,restriction endonuclease digestion, the mismatch repair activity willinclude mismatch detection, strand cutting and excision and strandresynthesis to fill the excision and ligation. When the modification isdetected in a recombination defective bacteria, e.g., E. coli strainDH10, the strand resynthesis and ligation activities may be omitted fromthe cell-free enzyme mixture. As used herein "mismatch repair activity"does not include the resynthesis and ligation activities, which may bepresent in the cell-free enzyme mixture but are not required in mostapplications.

In certain applications, e.g., to assay the effects of modifications ofthe recombinagenic oligonucleobase on its efficiency with plant ormammalian enzymes, it is preferred that the mismatch repair activity beprovided by the cell-free enzyme mixture. Detection by biochemical meansor in a host such as a MutS bacteria, e.g., NR9162, which lack mismatchrepair is preferred.

For certain applications, it is desirable to separate the complex oftarget DNA and recombinagenic oligonucleobase from the uncomplexedtarget DNA. Separation can be readily accomplished by introducing anaffinity ligand, e.g., a biotin, onto the recombinagenicoligonucleobase. In such applications, two cell-free enzyme mixtures canbe used, one before and one after the separation. The first mixtureshould contain only the strand transfer activity and the second needcontain only the mismatch repair activity.

The cell-free enzyme mixture can be obtained as a cell extract. Aprocedure of Li & Kelly can be used. Li., J. J., et alia., 1985, Mol.Cell. Biol. 5, 1238-1246. The Li & Kelly procedure is a "cytoplasmicextract." The cells are mechanically disrupted in hypotonic buffer andthe supernatant from centrifugation of 10 min. at 2,000×g and twice of15 min. at 12,000×g is used. Without limitation as to theory, it isbelieved that the physiological cellular location of the strand transferand mismatch repair enzymes is the nucleus but that during preparationthere is sufficient loss of these enzymes from the nucleus. Crudenuclear extracts made according to Dignam et al., 1983, Nucleic AcidResearch 11, 1475 are not preferred.

A cell-free enzyme mixture that lacks mismatch repair can be obtainedfrom extracts of mutant cells having the replication error phenotype.Umar et al., 1994, J. Biol. Chem. 269, 14367. The cell line LoVo hasdeleted both alleles of the human MutS homolog (MSH2) and is suitable asa source of strand transfer activity without mismatch repair activity.

In an alternative embodiment the cell-free enzyme mixture can be acomposition comprising recombinantly produced enzymes. The recombinantproduction of a defined enzyme allows for the addition of a known amountof the defined enzyme free of all other enzymes involved in the strandtransfer and mismatch repair. When a defined enzyme is added to anextract from a cell that is deficient in that enzyme the result is adefined enzyme mixture with regard to that enzyme. The production ofrecombinant Rad51 can be accomplished by the methods reported by Gupta,R. C., 1997, Proc. Natl. Acad. Sci. 94, 463-468.

5.2 The Recombinagenic Oligonucleobase

Recombinagenic oligonucleobases for use in a cell-free system can beconstructed according to the teaching of U.S. Pat. No. 5,565,350 andU.S. Pat No. 5,731,181. Additionally, recombinagenic oligonucleobasescan be made according to the following.

Definitions

The invention is to be understood in accordance with the followingdefinitions.

An oligonucleobase is a polymer of nucleobases, which polymer canhybridize by Watson-Crick base pairing to a DNA having the complementarysequence.

Nucleobases comprise a base, which is a purine, pyrimidine, or aderivative or analog thereof. Nucleobases include peptide nucleobases,the subunits of peptide nucleic acids, and morpholine nucleobases aswell as nucleobases that contain a pentosefuranosyl moiety, e.g., anoptionally substituted riboside or 2'-deoxyriboside. Nucleotides arepentosefuranosyl containing nucleobases that are linked byphosphodiesters. Other pentosefuranosyl containing nucleobases can belinked by substituted phosphodiesters, e.g., phosphorothioate ortriesterified phosphates.

A oligonucleobase compound has a single 5' and 3' end nucleobase, whichare the ultimate nucleobases of the polymer. Nucleobases are eitherdeoxyribo-type or ribo-type. Ribo-type nucleobases are pentosefuranosylcontaining nucleobases wherein the 2' carbon is a methylene substitutedwith a hydroxyl, substituted oxygen or a halogen. Deoxyribo-tapenucleobases are nucleobases other than ribo-type nucleobases and includeall nucleobases that do not contain a pentosefuranosyl moiety, e.g.,peptide nucleic acids.

An oligonucleobase strand generically includes regions or segments ofoligonucleobase compounds that are hybridized to substantially all ofthe nucleobases of a complementary strand of equal length. Anoligonucleobase strand has a 3' most (3' terminal) nucleobase and a 5'most (5' terminal) nucleobase. The 3' most nucleobase of a strandhybridizes to the 5' most nucleobase of the complementary strand. Twonucleobases of a strand are adjacent nucleobases if they are directlycovalently linked or if they hybridize to nucleobases of thecomplementary strand that are directly covalently linked. Anoligonucleobase strand may consist of linked nucleobases, wherein eachnucleobase of the strand is covalently linked to the nucleobasesadjacent to it. Alternatively a strand may be divided into two chainswhen two adjacent nucleobases are unlinked. The 5' (or 3') terminalnucleobase of a strand can be linked at its 5'-O (or 3'-) to a linkerwhich linker is further linked to a 3' (or 5') terminus of a secondoligonucleobase strand, which is complementary to the first strand,whereby the two strands form a single oligonucleobase compound. Thelinker can be an oligonucleotide, an oligonucleobase or other compound.The 5'-O and the 3'-O of a 5' end and 3' end nucleobase of anoligonucleobase compound can be substituted with a blocking group thatprotects the oligonucleobase strand. However, for example, closedcircular oligonucleotides do not contain 3' or 5' end nucleotides. Notethat when an oligonucleobase compound contains a divided strand the 3'and 5' end nucleobases are not the terminal nucleobases of a strand.

Conformation

The Duplex Mutational Vectors (DMV) are comprised of polymers ofnucleobases, which polymers hybridize, i.e., form Watson-Crick basepairs of purines and pyrimidines, to DNA having the appropriatesequence. Each DMV is divided into a first and a second strand of atleast 12 nucleobases and not more than 75 nucleobases. In a preferredembodiment the length of the strands are each between 20 and 50nucleobases. The strands contain regions that are complementary to eachother. In a preferred embodiment the two strands are complementary toeach other at every nucleobase except the nucleobases wherein the targetsequence and the desired sequence differ. At least two non-overlappingregions of at least 5 nucleobases are preferred.

Nucleobases contain a base, which is either a purine or a pyrimidine oranalog or derivative thereof. There are two types of nucleobases.Ribo-type nucleobases are ribonucleosides having a 2'-hydroxyl,substituted 2'-hydroxyl or 2'-halo-substituted ribose. All nucleobasesother than ribo-type nucleobases are deoxyribo-type nucleobases. Thus,deoxy-type nucleobases include peptide nucleobases.

In the embodiments wherein the strands are complementary to each otherat every nucleobase, the sequence of the first and second strandsconsists of at least two regions that are homologous to the target geneand one or more regions (the "mutator regions") that differ from thetarget gene and introduce the genetic change into the target gene. Themutator region is directly adjacent to homologous regions in both the 3'and 5' directions. In certain embodiments of the invention, the twohomologous regions are at least three nucleobases, or at least sixnucleobases or at least twelve nucleobases in length. The total lengthof all homologous regions is preferably at least 12 nucleobases and ispreferably 16 and more preferably 20 nucleobases to about 60 nucleobasesin length. Yet more preferably the total length of the homology andmutator regions together is between 25 and 45 nucleobases and mostpreferably between 30 and 45 nucleobases or about 35 to 40 nucleobases.Each homologous region can be between 8 and 30 nucleobases and morepreferably be between 8 and 15 nucleobases and most preferably be 12nucleobases long.

One or both strands of the DMV can optionally contain ribo-typenucleobases. In a preferred embodiment a first strand of the DMVconsists of ribo-type nucleobases only while the second strand consistsof deoxyribo-type nucleobases. In an alternative preferred embodimentthe second strand is divided into a first and second chain. The firstchain contains no ribo-type nucleobases and the nucleotides of the firststrand that are paired with nucleobases of first chain are ribo-typenucleobases. In an alternative embodiment the first strand consists of asingle segment of deoxyribo-type nucleobases interposed between twosegments of ribo-type nucleobases. In said alternative embodiment theinterposed segment contains the mutator region or, in the case of aHDMV, the intervening region is paired with the mutator region of thealternative strand.

Preferably the mutator region consists of 20 or fewer bases, morepreferably 6 or fewer bases and most preferably 3 or fewer bases. Themutator region can be of a length different than the length of thesequence that separates the regions of the target gene homology with thehomologous regions of the DMV so that an insertion or deletion of thetarget gene results. When the DMV is used to introduce a deletion in thetarget gene there is no base identifiable as within the mutator region.Rather, the mutation is effected by the juxtaposition of the twohomologous regions that are separated in the target gene. For thepurposes of the invention, the length of the mutator region of a DMVthat introduces a deletion in the target gene is deemed to be the lengthof the deletion. In one embodiment the mutator region is a deletion offrom 6 to 1 bases or more preferably from 3 to 1 bases. Multipleseparated mutations can be introduced by a single DMV, in which casethere are multiple mutator regions in the same DMV. Alternativelymultiple DMV can be used simultaneously to introduce multiple geneticchanges in a single gene or, alternatively to introduce genetic changesin multiple genes of the same cell. Herein the mutator region is alsotermed the heterologous region. When the different desired sequence isan insertion or deletion, the sequence of both strands have the sequenceof the different desired sequence.

The DMV is a single oligonucleobase compound (polymer) of between 24 and150 nucleobases. Accordingly the DMV contains a single 3' end and asingle 5' end. The first and the second strands can be linked covalentlyby nucleobases or by non-oligonucleobase linkers. In a preferredembodiment the 3' terminal nucleobase of each strand is protected from3' exonuclease attack. Such protection can be achieved by severaltechniques now known to these skilled in the art or by any technique tobe developed.

In one embodiment protection from 3'-exonuclease attack is achieved bylinking the 3' most (terminal) nucleobase of one strand with the 5' most(terminal) nucleobase of the alternative strand by a nuclease resistantcovalent linker, such as polyethylene glycol, poly-1,3-propanediol orpoly-1,4-butanediol. The length of various linkers suitable forconnecting two hybridized nucleic acid strands is understood by thoseskilled in the art. A polyethylene glycol linker having from six tothree ethylene units and terminal phosphoryl moieties is suitable.Durand, M. et al., 1990, Nucleic Acid Research 18, 6353; Ma, M. Y-X., etal.,1993, Nucleic Acids Res. 21, 2585-2589. A preferred alternativelinker is bis-phosphorylpropyl-trans-4,4'-stilbenedicarboxamide.Letsinger, R. L., et alia, 1994, J. Am. Chem. Soc. 116, 811-812;Letsinger, R. L. et alia, 1995, J. Am. Chem. Soc. 117, 7323-7328. Suchlinkers can be inserted into the DMV using conventional solid phasesynthesis. Alternatively, the strands of the DMV can be separatelysynthesized and then hybridized and the interstrand linkage formed usinga thiophoryl-containing stilbenedicarboxamide as described in patentpublication WO 97/05284, Feb. 13, 1997, to Letsinger R. L. et alia.

In a further alternative embodiment the linker can be a single strandoligonucleobase comprised of nuclease resistant nucleobases, e.g., a2'-O-methyl, 2'-O-allyl or 2'-F ribonucleotides. The tetraribonucleotidesequences TTTT, UUUU and UUCG and the trinucleotide sequences TTT, UUU,and UCG are particularly preferred nucleotide linkers.

In an alternative embodiment, 3'-exonuclease protection can be achievedby the modification of the 3' terminal nucleobase. If the 3' terminalnucleobase of a strand is a 3' end, then a steric protecting group canbe attached by esterification to the 3'-OH, the 2'-OH or to a 2' or 3'phosphate. A suitable protecting group is a 1,2-(ω-amino)-alkyldiol oralternatively a 1,2-hydroxymethyl-(ω-amino)-alkyl. Modifications thatcan be made include use of an alkene or branched alkane or alkene, andsubstitution of the ω-amino or replacement of the ω-amino with anω-hydroxyl. Other suitable protecting groups include a 3' endmethylphosphonate, Tidd, D. M., et alia, 1989, Br. J. Cancer, 60,343-350; and 3'-aminohexyl, Gamper H. G., et al., 1993, Nucleic AcidsRes., 21, 145-150. Alternatively, the 3' or 5' end hydroxyls can bederivatized by conjugation with a substituted phosphorus, e.g., amethylphosphonate or phosphorothioate.

In a yet further alternative embodiment the protection of the3'-terminal nucleobase can be achieved by making the 3'-most nucleobasesof the strand nuclease resistant nucleobases. Nuclease resistantnucleobases include peptide nucleic acid nucleobases and 2' substitutedribonucleotides. Suitable substituents include the substituents taughtby U.S. Pat. No. 5,731,181, and by U.S. Pat. No. 5,334,711 (Sproat),which are hereby incorporated by reference, and the substituents taughtby patent publications EP 629 387 and EP 679 657 (collectively, theMartin Applications), which are hereby incorporated by reference. Asused herein a 2' fluoro, chloro or bromo derivative of a ribonucleotideor a ribonucleotide having a substituted 2'-O as described in the MartinApplications or Sproat is termed a "2'-Substituted Ribonucleotide."Particular preferred embodiments of 2'-Substituted Ribonucleotides are2'-fluoro, 2'-methoxy, 2'-propyloxy, 2'-allyloxy, 2'-hydroxylethyloxy,2'-methoxyethyloxy, 2'-fluoropropyloxy and 2'-trifluoropropyloxysubstituted ribonucleotides. In more preferred embodiments of2'-Substituted Ribonucleotides are 2'-fluoro, 2'-methoxy,2'-methoxyethyloxy, and 2'-allyloxy substituted nucleotides.

The term "nuclease resistant ribonucleoside" encompasses including2'-Substituted Ribonucleotides and also all 2'-hydroxyl ribonucleosidesother than ribonucleotides, e.g., ribonucleotides linked bynon-phosphate or by substituted phosphodiesters. Nucleobase resistantdeoxyribonucleosides are defined analogously. In a preferred embodiment,the DMV preferably includes at least three and more preferably sixnuclease resistant ribonucleosides. In one preferred embodiment the CMVcontains only nuclease resistant ribonucleosides anddeoxyribonucleotides. In an alternative preferred embodiment, everyother ribonucleoside is nuclease resistant.

Each DMV has a single 3' end and a single 5' end. In one embodiment theends are the terminal nucleobases of a strand. In an alternativeembodiment, a strand is divided into two chains that are linkedcovalently through the alternative strand but not directly to eachother. In embodiments wherein a strand is divided into two chains, the3' and 5' ends are Watson-Crick base paired to adjacent nucleobases ofthe alternative strand. In such strands the 3' and 5' ends are notterminal nucleobases. A 3' end or 5' end that is not the terminalnucleobase of a strand can be optionally substituted with a stericprotector from nuclease activity as described above. In yet analternative embodiment, a terminal nucleobase of a strand is attached toa nucleobase that is not paired to a corresponding nucleobase of theopposite strand and is not a part of an interstrand linker. Suchembodiment has a single "hairpin" conformation with a 3' or 5'"overhang." The unpaired nucleobase and other components of the overhangare not regarded as a part of a strand. The overhang may includeself-hybridized nucleobases or non-nucleobase moieties, e.g., affinityligands or labels. In a particular preferred embodiment of DMV having a3' overhang, the strand containing the 5' nucleobase is composed ofdeoxy-type nucleobases only, which are paired with ribo-type nucleobaseof the opposite strand. In a yet further preferred embodiment of DMVhaving a 3' overhang, the sequence of the strand containing the 5' endnucleobase is the different, desired sequence and the sequence of thestrand having the overhang is the sequence of the target DNA.

A particularly preferred embodiment of the invention is a DMV whereinthe two strands are not fully complementary. Rather the sequence of onestrand comprises the sequence of the target DNA to be modified and thesequence of the alternative strand comprises the different, desiredsequence that the user intends to introduce in place of the targetsequence. It follows that the location where the target and desiredsequences differ, the bases of one strand are paired withnon-complementary bases in the other strand. Such DMV are termed hereinHeteroduplex Mutational Vectors (HDMV). In one preferred embodiment, thedesired sequence is the sequence of a chain of a divided strand. In asecond preferred embodiment, the desired sequence is found on a chain ora strand that contains no ribo-type nucleobases. In a more preferredembodiment, the desired sequence is the sequence of a chain of a dividedstrand, which chain contains no ribo-type nucleobases.

Internucleobase linkages

The linkage between the nucleobases of the strands of a DMV can be anylinkage that is compatible with the hybridization of the DMV to itstarget sequence. Such sequences include the conventional phosphodiesterlinkages found in natural nucleic acids. The organic solid phasesynthesis of oligonucleotides having such nucleotides is described inU.S. Pat. No. Re: 34,069.

Alternatively, the internucleobase linkages can be substitutedphosphodiesters, e.g., phosphorothioates, substituted phosphotriesters.Alternatively, non-phosphate, phosphorus-containing linkages can beused. U.S. Pat. No. 5,476,925 to Letsinger describes phosphoramidatelinkages. The 3'-phosphoramidate linkage (3'-NP(O⁻)(O)O-5') is wellsuited for use in DMV because it stabilizes hybridization compared to a5'-phosphoramidate. Non-phosphate linkages between nucleobases can alsobe used. U.S. Pat. No. 5,489,677 describes internucleobase linkageshaving adjacent N and O and methods of their synthesis. The linkage3'-ON(CH₃)CH₂ -5' (methylenemethylimmino) is a preferred embodiment.Other linkages suitable for use in DMV are described in U.S. Pat. No.5,731,181 to Kmiec. Nucleobases that lack a pentosefuranosyl moiety andare linked by peptide bonds can also be used in the invention.Oligonucleobases containing such so-called peptide nucleic acids (PNA)are described in U.S. Pat. No. 5,539,082 to Nielsen. Methods for makingPNA/nucleotide chimera are described in WO 95/14706.

5.3 Specific Uses

Heteroduplex Mutational Vectors of the invention and Non-chimericMutational Vectors of the invention can be used in any eukaryotic cellin the place of the prior art Chimeric Mutational Vectors. Patentpublication WO 97/41141 by Kmiec et al. teaches the use of ChimericMutational Vectors, ex vivo as do U.S. Pat. No. 5,565,350 and U.S. Pat.No. 5,731,181. Kren et al., 1998, Nature Medicine 4, 285 providesguidance for the use of Chimeric Mutational Vectors in vivo.

The recombinagenic oligonucleotides can be used in cell-free systems forseveral purposes, which will be apparent to those skilled in the art.Examples without limitation are as follows.

The effects of modification in the purity, chemistry, size and/orconformation of recombinagenic oligonucleotides can be rapidly andquantitatively tested in cell-free systems. The cell-free system has thefurther advantages that efficiency of recombination can be measuredindependently of the efficiency of delivery.

The cell-free system can be used to test compounds that are intended toinhibit or enhance the activity of the enzymes needed for chimeraplasty,in an alternative embodiment test for compounds that replace an enzymeof the mixture. Inhibitory compounds may be competitive ornon-competitive inhibitors that act directly on the enzymes involved.Alternatively, the inhibitors can act on the cell from which an extractis made to block the synthesis or accelerate the degradation of anenzyme. These compounds may act by inducing or suppressing the synthesisof the relevant enzymes or may act by inducing post-syntheticmodifications that activate or inactivate the relevant enzymes.

The cell-free system can be further used to test the relevance orparticular proteins to the mechanism of chimeraplasty. Such testing can,for example without limitation be performed by use of protein-specificmonoclonal antibodies to determine whether the protein in question isrelevant to chimeraplasty.

A further use of the cell-free system is the specific modification ofplasmid, or other isolated DNA molecules. In one embodiment of use forthis purpose, the recombinagenic oligonucleobase contains an affinityligand, such as biotin, that allows the separation of the complex withthe target DNA from the uncomplexed target DNA. The chimeraplastyreaction is, in this embodiment, performed using a separate strandtransfer step and a mismatch repair step. This embodiment can be used toincrease the proportion of modified DNA targets, so that non-selectablemodifications can be made without undue expenditure of material andeffort in screening. In one embodiment, the receptor for the affinityligand is bound to a solid phase particle so that the recombinagenicoligonucleobase/target DNA complex is attached to the particle. In thesecond stage of the reaction the mismatch repair activity results in themodification and release of the target DNA, whereby the supernatant ofthe second stage of the process is enriched for the modified plasmid.

6. EXAMPLES

Table I below shows the relative numbers of kanamycin and ampicillinresistant colonies using variants of Kany.y to correct a stop-codoncausing CG transversion in the kan resistance gene.

The following materials and methods were employed to obtain these data.

Cell-Free Extracts

HuH-7 (Nakabayashi, H., et al., 1982, Cancer Res. 42, 3858) cells aregrown in DMEM supplemented with 10% fetal bovine serum to mid log phase,about 5×10⁵ cells/ml. The cells are mechanically dislodged from thetissue culture flask and pelleted at 500×g. The pellet is washed inice-cold Hypotonic Buffer with sucrose (20 mM HEPES, pH 7.5, 5 mM KCl,1.5 mM MgCl₂, 1 mM DTT, 250 mM sucrose), washed in ice-cold HypotonicBuffer without sucrose and then resuspended in Hypotonic Buffer at6.5×10⁷ cells/ml and incubated on ice for 15 min. Thereafter the cellsare lysed using a Dounce homogenizer, 3-5 strokes, and thereafterincubated a further 45 min on ice. The lysate is cleared bycentrifugation at 10,000×g for 10 min. and the supernatant aliquoted andstored at -80° C. until use.

Reaction Conditions

The cell-free enzyme mixture, plasmid and DMV are reacted in a finalvolume of 50 μl. The reaction buffer is 20 mM Tris, pH 7.4, 15 mM MgCl₂,0.4 mM DTT, and 1.0 mM ATP. Plasmid, DMV and extract protein finalconcentrations are 20 μg/ml, 20 μg/ml and 600 μg/ml, respectively. Thereaction is run in 500 μl "Eppendorf" tubes. The tubes are prechilled onice and the reagents added and mixed except for the extract. The extractis then added and the reaction incubated 45 min at 37° C. The reactionis stopped by chloroform/phenol extraction. The nucleic acid isprecipitated with 10% (v/v) 3M sodium acetate, pH 4.8 and 2 volumes ofabsolute EtOH, at -20° C.

Bacterial Transformation

The precipitated, DMV-treated plasmid is dissolved and bacteria aretransformed by electroporation according to standard techniques. Afterelectroporation the bacteria are incubated for 1 hr in the absence ofantibiotic (kanamycin) and then for 4 hours in the presence of 20% ofthe selective level of antibiotic.

Analysis

The effectiveness of the DMV can be ascertained from the ratio of thekanamycin resistant colonies and the ampicillin resistant colonies,which is a measure of the recovery of the plasmid and the efficiency ofelectroporation. The ratio given in the table below is based on dataobtained after a 4 hour incubation with a sub-selective level ofkanamycin. Such selective incubation results in an increase in kan^(r)colonies of about 100 fold. The absolute frequencies, which have beencorrected for the pre-plating selection are reported.

DMV

The general structure of a Duplex Mutational Vector for the introductionof kanamycin resistance is given below. The intervening segment, 3'homology region, and 5' homology region are designated "I", "H-3'" and"H-5'", respectively. The interstrand linkers are designated "L". Anoptional chi site (5'-GCTGGTGG-3') and its complement are indicated as Xand X' respectively. The 3' and 5' mutator region are single nucleotidesindicated as M^(3') and M^(5'), respectively. Variant I is similar tothe Chimeric Mutational Vectors described in Cole-Strauss, 1996, Science273, 1386, and Kren, 1998, Nature Medicine 4, 285-290. Variant I isreferred to as Kany.y elsewhere in this specification. The symbol "--"for a feature of a variant indicates that the feature of the variant isthe same as variant I.

The above DMV causes a CG transversion that converts a TAG stop codoninto a TAC tyr codon. Note that the first strand of I lacks anexonuclease protected 3' terminus and that the second strand of I is adivided strand, the first chain of which is the desired, differentsequence. Variants IV and V are a Chimeric Mutational Vector and aNon-Chimeric Mutational Vector, respectively, having 3' terminiexonuclease protected by a nuclease resistant linker (2'OMe-U₄).Variants VIa and VIb are Chimeric Heteroduplex Mutational Vectors.Variant VIb is the variant in which the desired, different sequence isfound on the first chain, which chain consists of DNA-type nucleotidesonly.

The table below gives the activities of the variants relative to thevariant I in for a bacterial system and gives the frequency ofconversion to kan^(r) /10⁵ plasmids for a cell-free extract. Thebackground rates were negligible compared to the experimental valuesexcept for variant VIa in the cell-free system and bacterial systems andvariant VII in bacteria. The data reported for these variants arebackground corrected. Variants VIa and VII show low or absent activity.Each of variants Ill-V are superior in both systems to variant I, whichis of the type described in the scientific publications of Yoon,Cole-Strauss and Kren cited herein above. Variant VIII is the optimalchimera based on inference from these data.

The results shows an excellent correlation between activity in thecell-free extract and activity in the bacterial system. In particular,in both systems variants IV and VIb are superior to Kany.y and in bothsystems the Non Chimeric Mutational Vectors are active. The onlydisparity is variant VII, which contains solely deoxynucleotides.Variant VII is active in the cell-free extract but not the bacterialsystem. Deoxyoligonucleotides have also been found inactive ineukaryotic cells. Without limitation as to theory, applicants believethat the activity of variant VII in the cell-free system is due to thereduced amount of nucleases present in the system compared tocell-containing systems. In particular, applicants have found that a5'-end labeled 46 nt single strand DNA was not degraded (<1%) by thecell-free extract in a 10 min incubation at 37° C. incubation. A likeresult was obtained with a 46 bp 5' end labeled linear duplex DNAsubstrate. The reaction buffer was 2 mM ATP, 1 mM DTT, 25 mMTris-Acetate, pH 7.15, 5 mM Mg.

                                      TABLE 1                                     __________________________________________________________________________                                    kan.sup.r/                                                                 R.A.                                                                             10.sup.5 amp.sup.r                            DMV                                                                              M.sup.5'                                                                        M.sup.3'                                                                        H5' I   H3' L     X (X')                                                                            (bac)                                                                            cell-free                                     __________________________________________________________________________    I  C G 2'-OMe                                                                            DNA 2'-OMe                                                                            T.sub.4                                                                             None                                                                              1  6.0                                           II     ---     --                                                                                --                                                                                 --                                                                                  3.2hi†                                                                        1.4‡.                         III                                                                                  ---     2'-OMe                                                                                     --                                                                             1.6                                                                                   13                                       IV     ---     --                                                                                --                                                                                 2'-OMe-U.sub.4                                                                 --     10.0                                                                              50                                        V      DNA    --                                                                                 DNA                                                                               2'-OMe-U.sub.4                                                                  --     3.0                                                                                9.8                                      VIa                                                                                  ----                                                                                  --                                                                                --                                                                                 --                                                                                    0.06*                                                                            0.25                                       VIb                                                                                   --C                                                                                  --                                                                                --                                                                                 --                                                                                    7.5                                                                                10.8                                     VI     ---     ---                                                                               --                                                                                 T.sub.3                                                                               4.2                                                                                N.D.                                     VII                                                                                  DNA    --                                                                                 DNA                                                                               --                                                                                     4.4       ˜0                            VIII                                                                                  2'-OMe                                                                           2'-OMe                                                                            2'-OMe                                                                             2'-OMe-U.sub.4                                                                     --     N.D.                                                                              N.D.                                      __________________________________________________________________________     *Site Specific Rate                                                           †GCTGGTGG                                                              R.A. (bac) = relative activity (bacteria);                                    N.D. = Not Determined                                                         ‡Result from an independent experiment normalized to other dat

The sequences of DMV for the introduction of tetracycline resistance isgiven below: ##STR1##

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 4                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 84 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Other                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #CCCAGTCSTA    60GACTGGG CACAAGCTGG TGGTTTTCCA CCAGCTTGTG                     #                84TTTTC GCGC                                                 - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 68 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Other                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #TAGCGCGCGT    60GACTGGG CACAATTTTT TGTGCCCAGT CSTAGCCGAA                     #          68                                                                 - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 68 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Other                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #GGAAGCGCGT    60GCCAGTC ACTATTTTTA TAGTGACTGG CAATGCTGTC                     #          68                                                                 - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 68 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Other                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #CCTAGCGCGT    60GGTTATG CCGGTTTTTA CCGGCATAAC CAAGCCTATG                     #          68                                                                 __________________________________________________________________________

We claim:
 1. A method of transforming a target DNA sequence into adifferent, desired sequence in a eukaryotic cell that comprises (A)administering to the cell a duplex mutational vector comprising:a. afirst oligonucleobase strand of at least 12 linked nucleobases and notmore than 75 linked nucleobases, which strand has a first terminalnucleobase and a second terminal nucleobase; b. a second oligonucleobasestrand having a 3' most nucleobase and a 5' most nucleobase and having anumber of nucleobases equal to the first strand, which second strand isoptionally divided into a first chain and a second chain; and c. a 3'end nucleobase and a 5' end nucleobase; in whichi. the 3' most and 5'most nucleobases of the second strand are Watson-Crick base paired tothe first terminal and the second terminal nucleobase of the firststrand, respectively, ii. said 3' most nucleobase and said secondterminal nucleobase are protected from 3' exonuclease attack, and iii.the second strand contains at least two non-overlapping regions of atleast 5 contiguous nucleobases that are Watson-Crick base paired tonucleobases of the first strand;provided that there are not more thantwo contiguous Watson-Crick base pairs comprised of a ribo-type and adeoxyribo-type nucleobase; and (B) detecting DNA from or in the cell orthe progeny thereof having the different, desired sequence.
 2. Themethod of claim 1, wherein each nucleobase of the first strand isWatson-Crick paired to a complementary nucleobase of the second strand.3. The method of claim 1, wherein the sequence of the first strandcomprises the sequence of the different, desired sequence.
 4. The methodof claim 1, wherein the first terminal nucleobase and the 3' mostnucleobase are linked by a linker comprising a moiety selected from thegroup consisting of 2'-methoxy-uridine, 2'-allyloxy-uridine,2'-fluoro-uridine, 2'-methoxy-thymidine, 2'-allyloxy-thymidine,2'-fluoro-thymidine, polyethylene glycol, andtrans-4,4'-stilbenecarboxamide.
 5. The method of claim 1, wherein thesecond terminal nucleobase and the 5' most nucleobase are linked by alinker comprising a moiety selected from the group consisting of2'-methoxy-uridine, 2'-allyloxy-uridine, 2'-fluoro-uridine,2'-methoxy-thymidine, 2'-allyloxy-thymidine, 2'-fluoro-thymidine,polyethylene glycol, and trans-4,4'-stilbenecarboxamide.
 6. The methodof claim 1, wherein the second strand is comprised of a first chain anda second chain and the first chain contains no ribo-type nucleobases. 7.The method of claim 6, wherein each nucleobase of the first strand isWatson-Crick paired to a complementary nucleobase of the second strand.8. The method of claim 6, wherein the sequence of the different, desiredsequence is the sequence of the first chain.
 9. The method of claim 1,wherein the first chain comprises the 5' end nucleobase.
 10. The methodof claim 1, wherein the first chain comprises the 3' end nucleobase.